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Male contraceptive options and infertility treatments are limited. To help overcome these limitations we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in our automated high-throughput screening platform and provide the dataset, which allows direct side-by-side comparisons of compound effects and assays using live human spermatozoa.

Despite recent advances in male reproductive health research, there remain many elements of male infertility where our understanding is incomplete. Consequently, diagnostic tools and treatments for men with sperm dysfunction, other than medically assisted reproduction, are limited. On the other hand, the gaps in our knowledge of the mechanisms which underpin sperm function have hampered the development of male non-hormonal contraceptives. The study of mature spermatozoa is inherently difficult. They are a unique and highly specialised cell type which does not actively transcribe or translate proteins and cannot be cultured for long periods of time or matured in vitro. One large-scale approach to both increasing the understanding of sperm function and the discovery and development of compounds that can modulate sperm function is to directly observe responses to compounds with phenotypic screening techniques. These target agnostic approaches can be developed into high-throughput screening platforms with the potential to drastically increase advances in the field. Here, we discuss the rationale and development of high-throughput phenotypic screening platforms for mature human spermatozoa and the multiple potential applications these present, as well as the current limitations and leaps in our understanding and the capabilities needed to overcome them. Further development and use of these technologies could lead to the identification of compounds which positively or negatively affect sperm cell motility or function or novel platforms for toxicology or environmental chemical testing among other applications. Ultimately, each of these potential applications is also likely to increase the understanding within the field of sperm biology.

How morphogenetic movements are robustly coordinated in space and time is a fundamental open question in biology. We study this question using the wing ofDrosophila melanogaster, an epithelial tissue that undergoes large-scale tissue flows during pupal stages. We showed previously (Etournay et al., 2015) that pupal wing morphogenesis involves both cellular behaviors that allow relaxation of mechanical tissue stress, as well as cellular behaviors that appear to be actively patterned. The core planar cell polarity (PCP) pathway influences morphogenetic cell movements in many other contexts, which suggests that it could globally pattern active cellular behaviors during pupal wing morphogenesis. We show here, however, that this is not the case: there is no significant phenotype on the cellular dynamics underlying pupal morphogenesis in mutants of core PCP. Furthermore, using laser ablation experiments, coupled with a rheological model to describe the dynamics of the response to laser ablation, we conclude that while core PCP mutations affect the fast timescale response to laser ablation, they do not affect overall tissue mechanics. In conclusion, our work shows that cellular dynamics and tissue shape changes duringDrosophilapupal wing morphogenesis are independent of one potential chemical guiding cue, core PCP.

There is a need for non-hormonal contraceptives. One area that needs further investigation is the development of male contraceptives. Comparatively little is understood about potential drug targets in men to achieve a reversible contraceptive effect. In this article, we review the need for male contraceptives and some thoughts around the characteristics of a male contraceptive and the potential development pathway. We then discuss different potential approaches to discovering male contraceptives and then highlight potential targets that have been discussed in the literature.

STUDY QUESTION Can a high-throughput screening (HTS) platform facilitate male fertility drug discovery? SUMMARY ANSWER An HTS platform identified a large number of compounds that enhanced sperm motility. WHAT IS KNOWN ALREADY Several efforts to find small molecules modulating sperm function have been performed but none have used high-throughput technology. STUDY DESIGN, SIZE, DURATION Healthy donor semen samples were used and samples were pooled (3–5 donors per pool). Primary screening was performed singly; dose–response screening was performed in duplicate (using independent donor pools). PARTICIPANTS/MATERIALS, SETTING, METHODS Spermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 μM) to identify those compounds with enhancing effects on motility. Approximately 17 000 compounds from the libraries, ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box, were screened. Dose–response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a university setting. MAIN RESULTS AND THE ROLE OF CHANCE From our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to dimethylsulphoxide-treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified, in particular PDE10A inhibitors as well as number of compounds not previously known to enhance human sperm motility, such as those related to GABA signalling. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Although this approach provides data about the activity of the compound, it is only a starting point. For example, further substantive experiments are necessary to provide a more comprehensive picture of each compound’s activity, the effect on the kinetics of the cell populations and subpopulations, and their potential mechanisms of action. Compounds have been tested with prepared donor spermatozoa, incubated under non-capacitating conditions, and only incubated with compounds for a relatively short period of time. Therefore, the effect of compounds under different conditions, for example in whole semen, for longer incubation times, or using samples from patient groups, may be different and require further study. All experiments were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS This phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential compounds as promising start-point for a medicinal chemistry programme for potential enhancement of male fertility. Moreover, with disclosure of the results of screening, we present a substantial resource to inform further work in the field. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Bill and Melinda Gates Foundation, Scottish Funding Council and Scottish Universities Life Science Alliance. C.L.R.B. is Editor for RBMO. C.L.R.B. receives funding from Chief Scientists Office (Scotland), ESHRE and Genus PLC, consulting fees from Exscientia and lecture fees from Cooper Surgical and Ferring. S.M.d.S. is an Associate Editor of Human Reproduction, and an Associate Editor of Reproduction and Fertility. S.M.d.S. receives funding from Cooper Surgical and British Dietetic Society. No other authors declared a COI.

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology.

There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology.